CN116554328B - A single-chain antibody, CAR, CAR-NK cell targeting TRBV12 and its application - Google Patents

Abstract

The application provides a single-chain antibody targeting TRBV12, a CAR-NK cell and application, wherein the single-chain antibody targeting TRBV12 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three complementarity determining regions consisting of CDR-H1, CDR-H2 and CDR-H3, and the light chain variable region comprises three complementarity determining regions consisting of CDR-L1, CDR-L2 and CDR-L3; the amino acid sequence of the CDR-H1 is shown as SEQ ID NO. 2, the amino acid sequence of the CDR-H2 is shown as SEQ ID NO. 3, and the amino acid sequence of the CDR-H3 is shown as SEQ ID NO. 4; the amino acid sequence of CDR-L1 is shown as SEQ ID NO. 5, the amino acid sequence of CDR-L2 is shown as SEQ ID NO. 6, and the amino acid sequence of CDR-L3 is shown as SEQ ID NO. 7.

Description

A single-chain antibody, CAR, CAR-NK cell targeting TRBV12 and its application

Technical field

The present invention relates to the field of biotechnology, and specifically relates to a CAR-NK cell based on a single-chain antibody targeting TRBV12 and its preparation and application.

Background technique

T-cell malignancies are a widespread and heterogeneous group of diseases, each characterized by clonal proliferation of dysfunctional T cells at different stages of development and often with a poor prognosis. T-cell malignancies include T-cell acute lymphoblastic leukemia (T-ALL), cutaneous and peripheral T-cell lymphomas (CTCL and PTCL), and adult T-cell leukemia (ATL). T-cell malignant tumors progress rapidly and can quickly infiltrate into lymph nodes, liver, spleen, central nervous system and other tissues and organs in a short period of time. Currently, there is a lack of effective treatments. Although immunotherapy has achieved certain efficacy in the treatment of cancer through the use of monoclonal antibodies, checkpoint inhibitors, bispecific T-cell engagers, and chimeric antigen receptor (CAR) T cells, it has only been observed in T-cell diseases. to a limited response. Therefore, the treatment of malignant T-cell tumors is a major problem currently faced in clinical practice.

Currently, cell therapy in the treatment of T-cell malignant tumors mainly faces two limitations: First, currently CAR-T cell therapy has achieved good results in relapsed/refractory B-cell malignant tumors, with the broad-spectrum target CD19 CAR- T is a typical representative and has made great achievements in the treatment of hematoma. Judging from clinical data, the depletion of normal B cells is well tolerated by patients and is considered an acceptable side effect. However, although targeting a broad range of T cell antigens can control T cell tumors, it will lead to severe immune function destruction accompanied by depletion of normal T cells. Currently, a few studies use CD7 or CD5 as therapeutic targets for T cell malignancies. Since CD7/CD5 are also expressed on the surface of normal T cells, this broad targeting of T cell antigens causes severe T cell aplasia and immune function damage. Therefore, unlike B-cell tumors, for T-cell malignancies, it is necessary to seek a target that does not rely on a broad range of T-cell antigens but targets specific tumor cell populations, thereby retaining normal T cells and avoiding severe immune destruction as a treatment for T-cell malignancies. A major problem in tumors. Second, as CAR-T is more and more widely used in clinical applications, it has also shown certain drawbacks, the most important of which are cytokine release syndrome and neurotoxicity, which also greatly affects the therapeutic effect of CAR-T. In addition, the acquisition of autologous cells brings challenges to the clinical application of CAR-T cells. On the one hand, some patients cannot obtain enough high-quality T cells due to the impact of the disease; on the other hand, it takes a certain amount of time to prepare cells, which prevents some patients from receiving timely treatment. Therefore, choosing an immune cell therapy with low immunogenicity and realizing allogeneic reinfusion to replace traditional CAR-T cells has become a key scientific issue that needs to be further solved.

αβ T cell receptor (TCR) is a transmembrane heterodimer composed of two peptide chains α and β. It is responsible for specifically recognizing antigen peptides that bind to MHC (major histocompatibility complex) and activating T cells. cells generate subsequent immune responses. TCR is expressed on both normal T cells and T cell tumors. The TCR beta chain (TRB) germline locus consists of 68 variable (V) gene segments, as well as 2 diversity (D), 13 junction (J) and Composed of 2 constant (C) gene segments. The TCRβ chain variable gene (TRBV) segments are divided into 30 TRBV families based on nucleotide sequence similarity. During the development of T cells, V, D, and J gene segments undergo rearrangement, resulting in continuous VDJ transcripts expressing a unique TCRβ chain on the surface of each T cell, accounting for 1% to 1% of the total number of T cells in normal human peripheral blood. 5%. In T cell malignancies, clonal malignant proliferation of T cells occurs and only one type of TRBV is expressed, which provides a potential opportunity to selectively eliminate malignant clonal T cells while retaining most normal T cells. Some studies have used animal models to confirm that the loss of T cells expressing a single TRBV does not affect the body's normal immune response. Therefore, this study started from the problem of target selection of T cell malignant tumors and established an anti-TRBV12 chimeric antigen receptor that can selectively kill TRBV12(+) tumor cells while sparing other TRBV12(-) normal T cells. . Compared with targeting a wide range of T cell antigens, it avoids T cell regeneration caused by treatment and retains the body's normal immune function.

NK cells, or natural killer cells, are the first line of defense of the human immune system and can actively identify and kill viruses that invade the body and cells infected by viruses. In contrast to T cells with an extensively clonally rearranged repertoire of TCRs, NK cell activating or inhibitory signals are mediated by germline-encoded receptors such as NKG2D or KIR (Ig-like receptors) and CD94-NKG2 heterodimers, except In addition to direct cytotoxicity using granzymes and perforin, triggered NK cells can also lead to target cell destruction by stimulating the production of inflammatory cytokines. Loss of TCR significantly reduces the risk of GvHD compared to T cells, allowing the use of allogeneic CAR-NK cells for reinfusion. In addition, compared with CAR-T cells, CAR-NK cells are less likely to induce cytokine release syndrome (CRS) and have higher safety. Secondly, compared with using autologous T cells to prepare CAR-T, allogeneic CAR-NK cells can not only overcome the risk of malignant tumor cell contamination of the final product, but also reduce the cost and time required for the preparation of autologous products, making it easier to use at any time.

Contents of the invention

Based on the above background, there is currently no effective treatment method for T cell malignant tumors. The present invention provides a human-based single-chain antibody targeting TRBV12, and uses the single-chain antibody as an antigen-binding domain to construct a chimeric Antigen receptor CAR, expressed by CAR-NK cells, can effectively eliminate TRBV12(+) T cell malignancies.

The first object of the present invention is to provide a single-chain antibody targeting TRBV12, including a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes CDR-H1, CDR-H2 and CDR- H3 consists of three complementarity determining regions, and the light chain variable region includes three complementarity determining regions consisting of CDR-L1, CDR-L2 and CDR-L3; the amino acid sequence of CDR-H1 is shown in SEQ IDNO: 2, CDR- The amino acid sequence of H2 is shown in SEQ ID NO:3, the amino acid sequence of CDR-H3 is shown in SEQ ID NO:4; the amino acid sequence of CDR-L1 is shown in SEQ ID NO:5, and the amino acid sequence of CDR-L2 is shown in SEQ ID NO:6 is shown, and the amino acid sequence of CDR-L3 is shown as SEQ ID NO:7.

SEQ ID NO:2:NFRMH;

SEQ ID NO:3:YISSGSSTIYYADTMKG;

SEQ ID NO:4:RGEGAMDY;

SEQ ID NO:5:RAGSSVNYIY;

SEQ ID NO:6:YTSNLAP;

SEQ ID NO:7:QRFTSSPFT;

Further, the full-length amino acid sequence of the antibody is shown in SEQ ID NO: 1.

SEQ ID NO:1：

DVQLVESGGGLVQPKGSRKLSCAASGFTFSNFRMHWVRRAPGKGLEMVAYISSGSSTIYYADTMKGRFTISRDNPKNTLFLQMTSLRSEDTAMYYCARRGEGAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSENVLTQSPATMSASSLGIKVTMSCRAGSSVNYIYWYQQKSDASPKLWIYYTSNLAPGVPTRFSGSGSGNSLSLLTISSMEGE DAATYYCQQFTSSPFTFGQGTKLEIK.

The second object of the present invention is to provide an isolated nucleic acid encoding the above-mentioned single-chain antibody.

Further, its nucleotide sequence is shown in SEQ ID NO:8.

SEQ ID NO:8:

GACGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCAAGGGCAGCAGGAAGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAACTTCAGGATGCACTGGGTGAGGAGGGCCCCGGCAAGGGCCTGGAGATGGTGGCCTACATCAGCAGCGGCAGCAGCACCATCTACTACGCCGACACCATGAAGGGCAGGTTCACCATCAGCAGGGACAACCCCAAGAACACCCTGTTCCTGCAGATGACCAG CCTGAGGAGCGAGGACACCGCCATGTACTACTGCGCCAGGAGGGGCGAGGGCGCCATGGACTACTGGGGCCAGGGCACCAGCGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCAGCGAGAACGTGCTGACCCAGAGCCCCGCCACCATGAGCGCCAGCCTGGGCATCAAGGTGACCATGAGCTGCAGGGCCGGCAGCAGCGTGAACTACATCTACTGGTACCAGCAGAAGAGC GACGCCAGCCCCAAGCTGTGGATCTACTACACCAGCAACCTGGCCCCCGGCGTGCCCACCAGGTTCAGCGGCAGCGGCAGCGGCAACAGCCTGAGCCTGACATCAGCAGCATGGAGGGCGAGGACGCCGCCACCTACTACTGCCAGAGGTTCACCAGCAGCCCCTTCACCTTCGGCCAGGGCACCAAGCTGGAGATCAAG.

The third object of the present invention is to provide an expression vector or host cell containing the above-mentioned isolated nucleic acid.

The fourth object of the present invention is to provide a chimeric antigen receptor CAR containing the above-mentioned single chain antibody, characterized in that: the chimeric antigen receptor CAR also includes a transmembrane domain and a costimulatory signaling region.

Further, the transmembrane domain is selected from one of CD3ζ, CD8, CD28, NKG2D, 2B4, and DNAM1;

The costimulatory signaling region is selected from CD3ζ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD66d, CD2, CD4, CD5, CD28 family, DAP10, DAP12, NKp44, NKG2D, NKp46, NKp30, TNFR family or one or more combinations from the SLAM receptor family.

Further, the CD28 family is CD28 or ICOS; the TNFR family is 4-1BB, OX40 or CD27; and the SLAM receptor family is 2B4.

The fifth object of the present invention is to provide a CAR-NK cell expressing the above-mentioned chimeric antigen receptor CAR.

The sixth object of the present invention provides a method for preparing CAR-NK cells, which includes the following steps:

Isolate and culture NK cells from healthy people;

The PB transposon system is used to electroporate NK cells; the PB transposon system expresses anti-TRBV12 CAR;

Detect the expression efficiency of CAR in NK cells after electroporation, and further expand the culture.

The seventh object of the present invention is to provide a method for preparing anti-TRBV12(+) T cell malignant tumors or TRBV12(+) T cell proliferative diseases by using the above-mentioned single chain antibodies, chimeric antigen receptor CAR, or CAR-NK cells. Applications in medicines or products.

Beneficial technical effects achieved by the present invention: Aiming at the target of T cell tumors, the present invention uses antibodies targeting TRBV12 to construct a mutant library, and combines phage display technology to screen out mutant antibodies, that is, single-chain antibodies targeting TRBV12. The single-chain antibody was used as the scFV part of the chimeric antigen receptor CAR. By integrating the chimeric antigen receptor CAR into NK cells, CAR-NK cells are prepared, and their efficacy against T cell malignancies has been verified. It not only provides treatment for TRBV12(+) T cell tumors, but also uses TRBV12 as an entry point to continue to develop CAR-NK cells targeting other TRBV families, further laying a research foundation for clinically conquering T cell malignant tumors. Secondly, the present invention also provides a CAR-NK cell targeting TRBV12. The CAR-NK cell can express a chimeric antigen receptor CAR, wherein the chimeric antigen receptor CAR includes an antigen-binding domain and a transmembrane Domains and costimulatory signaling regions. The antigen domain is a single-chain antibody targeting TRBV12, which can specifically bind to TRBV12(+) T cells and activate the NK cells through the transmembrane domain and costimulatory signaling region; the CAR-NK cells have The following advantages: 1. Strong targeting, with the potential to eliminate malignant tumor cells while retaining normal T cells; 2. Small side effects. Compared with CAR-T, CAR-NK can not only reduce the risk of cytokine storm but also Avoid the risk of malignant cell contamination in the final product; 3. Good versatility, using NK cells to effectively reduce GvHD, and reduce preparation time and cost; 4. Innovative concept, using single-chain antibodies targeting TRBV12 as the target recognition region of CAR , achieving functional breakthroughs through technology upgrades.

Description of the drawings

Figure 1 shows the affinity of TRBV12-His antibodies to cells expressing TRBV12(+); where A indicates that the commercial TRBV12-biolegend antibody and the TRBV12-His antibody of the present application respectively recognize TRBV12(+) in the normal peripheral blood of two donors. The ability of cells; B represents the ability of TRBV12-His antibody and commercial TRBV12-biolegend antibody to recognize Jurkat cells endogenously expressing TRBV12; C represents the ability of TRBV12-His antibody and commercial TRBV12-biolegend antibody to recognize normal peripheral blood respectively. Statistical chart showing the recognition ability of TRBV12(+) cells and the recognition ability of Jurkat cells endogenously expressing TRBV12;

Figure 2 shows the PB transposon DNA vector expressing anti-TRBV12 CAR;

Figure 3 shows the transposase DNA vector expressing PiggyBac;

Figure 4 shows the detection of CAR-NK cell expression efficiency and NK cell phenotype; A represents the detection of CAR expression efficiency before and after CAR-NK cell sorting; B represents the detection of CAR-NK cell expression before and after sorting. Statistical analysis of CAR expression efficiency; C represents the expression of NK surface molecules on the surface of CAR-NK cells after 14 days of continued culture after sorting; D represents the statistical graph of the expression of NK surface molecules on the surface of CAR-NK cells;

Figure 5 shows the killing detection of (TRBV12-His) CAR-NK and (α-TRBV12) CAR-NK cells against Jurkat cells endogenously expressing TR BV12; where A indicates that the CAR-NK cells and target cells were treated in different The lysis of target cells after co-culture with effective target ratio. B and C represent the expression of cytokines TNF-α and IFN-γ in the supernatant;

Figure 6 shows the killing detection of (TRBV12-His) CAR-NK and (α-TRBV12) CAR-NK cells against CCRF cells overexpressing TRBV12 (CCRF-TRBV12). A indicates that the CAR-NK cells and target cells were used in different The lysis of target cells after co-culture with effective target ratio. B and C represent the expression of cytokines TNF-α and IFN-γ in the supernatant.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments. The following examples are only used to more clearly illustrate the technical solutions of the present invention, but cannot be used to limit the scope of the present invention.

The patent of the present invention will be further described below in conjunction with the accompanying drawings and examples.

Example 1 Screening of human heavy chain antibodies targeting TRBV12

The human heavy chain antibody targeting TRBV12 is a mutant library constructed based on the antibody sequence shown in SEQ ID NO: 9, and then the mutant antibody sequence is screened from the mutant antibody library using phage display technology. Finally, a single-chain antibody targeting TRBV12 was screened, and the amino acid sequence is shown in SEQ ID NO: 1. The specific process is as follows:

SEQ ID NO:9: DVQLVESGGGLVQPGGSRKLSCAASGFTFSNFGMHWVR QAPGKGLEWVAYISSGSSTIYYADTLKGRFTISRDNPKNTLFLQMTSLRSEDTAMYYCARRGEGAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSENVLTQSPAIMSASLGEKVTMSCRASSSVNYIYWYQQKSDASPKLWIYYTSNLAPGVPTRFSGS GSGNSYSLTISSMEGEDAATYYCQQFTSSPFTFGQGTKLEIK.

① Based on the antibody sequence shown in SEQ ID NO: 9, use error-prone PCR technology to prepare a PCR reaction system to amplify the target fragment; conduct 2% agarose gel electrophoresis analysis of the PCR product, and cut out about 500 bp of the target fragment Band, use a gel recovery column to recover the amplification product of the target fragment. ③Use SfiⅠ and NotⅠ to double-digest the pCANTAB5E vector and antibody genes. Recover the digested fragments from the previous step by agarose gel electrophoresis. Combine 1.5ug of vector, 0.5ug of antibody, 2μL of T4 ligase, 10μL of 10xbuffer, add water to 100μL, ligate on a PCR machine at 16°C overnight, and ligate the antibody gene into the digested pCANTAB5E vector. ④Add 5uL ligation product to 100uL TG1 competent cell, place it on ice to pre-cool, and transfer it to the pre-cooled electroporation cup; adjust the electroporation instrument voltage to 2.5KV and shock time 5ms. After electroshock, quickly add 0.9ml culture medium, 37 Incubate at ℃ for 2 hours with shaking; take 10uL gradient dilution and spread it on the SOBAG plate to calculate the storage volume. ⑤ Inoculate 1.5 mL of the antibody library into 300 mL of culture medium, incubate with shaking at 37°C for about 1.5 hours, add helper phage (M13K07) in 5 times the volume, and incubate with shaking at 37°C for about 1 hour; centrifuge at 4000 rpm at 15°C for 15 min, and remove the culture medium ;Add 200mL culture medium (100μg/mL Amp, 50μg/mL Kan) to resuspend the bacteria and culture at 37℃ for 2h; centrifuge at 10000rpm for 20min to remove the precipitate; add 40mL PEG/NaCl to precipitate the phage in the supernatant and keep on ice overnight; centrifuge at 10000rpm for 20min and remove Supernatant; resuspend the phage in 0.6 mL culture medium and set aside at 4°C. The obtained phages were serially diluted, infected with TG1 bacteria, spread on SOBAG plates, and the phage library titer was calculated by counting colonies. ⑥His Bind Resin was used to bind the antigen protein, and antibodies were selected from the phage display antibody library. Finally, a single-chain antibody targeting TRBV12 was successfully screened. Subsequent functional experiments showed that the mutant antibody had a greater killing effect than the original antibody. .

Example 2 Affinity verification of TRBV12-targeting human heavy chain antibodies for TRBV12(+) T cells

First, the screened single-chain antibody, such as the DNA sequence shown in SEQ ID NO:8, was used as a template to synthesize cDNA, and the C-terminal human IgG1 FC was connected to clone the fusion into the PCDH-EF1α vector. The plasmid was further transfected into 293T cells and the antibody (TRBV12-His) was purified using His column.

Since TRBV12-expressing T cells account for a small proportion in the population, we isolated peripheral blood T lymphocytes from 2 healthy adults, and used commercial TRBV12 antibodies (biolegend) and our screened antibodies (TRBV12-His) to combine with 2 donors at the same time. body's normal T lymphocytes. The results are shown in Figure 1, A and C. The proportion of T cells recognized by these antibodies is similar to that of commercial antibodies (biolegend). Jurkat cells that naturally express TRBV12 were further used to verify the activity of the antibody. The results were still consistent with the detection efficiency of commercial TRBV12 antibodies (biolegend), as shown in B and C in Figure 1, proving that the screened antibodies can recognize the target TRBV12 well.

Example 3 Construction of PB transposon system expressing anti-TRBV12 CAR structure

The single-chain antibody obtained above is combined with the CD8 transmembrane domain such as SEQ ID NO: 10 and the CD28-CD3ζ costimulatory domain such as SEQ ID NO: 11 to form a CAR structure targeting TRBV12, such as SEQ ID NO: 12. Connected to the vector plasmid PUC between the EcoRI and SalI restriction sites, it was named PUC-EF1α-#3, as shown in Figure 2. In addition, the transposase part was connected between the SFiI restriction sites of the PUC vector and named PUC-CMV-PiggyBac, as shown in Figure 3.

SEQ ID NO:10

ACCACCACCCCCGCCCCCCGCCCCCCCACCCCCGCCCCCACCATCGCCAGCCAGCCCCTGAGCCTGCGCCCCGAGGCCTGCCGCCCCGCCGCCGGCGGCCGCCGTGCACACCCGCGGCCTGGACTTCGCCTGCGACATCTACATCTGGGCCCCCCTGGCCGGCACCTGCGGCGTGCTGCTGCTGAGCCTGGTGATCACC.

SEQ ID NO:11

AAGCCGGCCGCAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGCGCCCCGTGCAGACCACCCAGGAGGAGGACGGCTGCAGCTGCCGCTTCCCCGAGGAGGAGGAGGGCGGCTGCGAGCTGCGCGTGAAGTTCAGCCGCAGCGCCGACGCCCCCGCTACCAGCAGGGCCAGAACCAGCTGTACAACGAGCTGAACCTGGGCCGCCGCGAGGAGTACGACGTGCTGGACAAGCGCCGCGGCCGCGACCCCG AGATGGGCGGCAAGCCCCAGCGCCGCAAGAACCCCCAGGAGGGCCTGTACAACGAGCTGCAGAAGGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGCGCCGCCGCGGCAAGGGCCACGACGGCCTGTACCAGGGCCTGAGCACCGCCACCAAGGACACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCCCGC.

SEQ ID NO:12

ATGGCCCTGCCCGTGACCGCCCTGCTGCTGCCCCTGGCCCTGCTGCTGCACGCCGCCCGCCCCGACGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCAAGGGCAGCAGGAAGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAACTTCAGGATGCACTGGGTGAGGAGGGCCCCGGCAAGGGCCTGGAGATGGTGGCCTACATCAGCAGCGGCAGCAGCACCATCTACTACGCCGACACCAT GAAGGGCAGGTTCACCATCAGCAGGGACAACCCCAAGAACACCCTGTTCCTGCAGATGACCAGCCTGAGGAGCGAGGACACCGCCATGTACTACTGCGCCAGGAGGGGCGAGGGCGCCATGGACTACTGGGGCCAGGGCACCAGCGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGAGAACGTGCTGACCCAGAGCCCCGCCACCATGAGCGCCAGCCTGGGGCATCAAGGTG ACCATGAGCTGCAGGGCCGGCAGCAGCGTGAACTACATCTACTGGTACCAGCAGAAGAGCGACGCCAGCCCCAAGCTGTGGATCTACTACACCAGCAACCTGGCCCCCGGCGTGCCCACCAGGTTCAGCGGCAGCGGCAGCAGCAGCCTGAGCCTGACCATCAGCAGCATGGAGGGCGAGGACGCCGCCACCTACTACTGCCAGAGGTTCACCAGCAGCCCCTTCACCTTCGGCCAGGGCACCAAGCTGGAT CAAGACCACCACCCCCGCCCCCCGCCCCCCCACCCCCGCCCCCACCATCGCCAGCCAGCCCCTGAGCCTGCGCCCCGAGGCCTGCCGCCCCGCCGCCGGCGGCCGCCGTGCACACCCGCGGCCTGGACTTCGCCTGCGACATCTACATCTGGGCCCCCCTGGCCGGCACCTGCGGCGTGCTGCTGCTGAGCCTGGTGATCACCAAGCGCGGCCGCAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGCGCCCCGTGCAGACCACCCAGG AGGAGGACGGCTGCAGCTGCCGCTTCCCCGAGGAGGAGGAGGGCGGCTGCGAGCTGCGCGTGAAGTTCAGCCGCAGCGCCGACGCCCCCGCCTACCAGCAGGGCCAGAACCAGCTGTACAACGAGCTGAACCTGGGCCGCCGCGAGGAGTACGACGTGCTGGACAAGCGCCGCGGCCGCGACCCCGAGATGGGCGGCAAGCCCCAGCGCCGCAAGAACCCCCAGGAGGGCCTGTACAACGAGCTGCAGAAGGACAAGA TGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGCGCCGCCGCGGCAAGGGCCACGACGGCCTGTACCAGGGCCTGAGCACCGCCACCAAGGACACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCCCGC.

Example 4 Preparation of CAR-NK cells targeting TRBV12

1.NK cell culture

Centrifuge the fresh blood at 2000 rpm for 10 minutes, take the upper layer of light yellow plasma into a 50ml centrifuge tube, inactivate it in a 56°C water bath for 30 minutes, then centrifuge at 400g for 10 minutes to remove the precipitate, transfer the upper layer of plasma to a new 50ml centrifuge tube and store it in a 4°C refrigerator For later use, dilute the lower blood cells with physiological saline 1:1 and mix well. First add 15ml of human peripheral blood lymphocyte separation solution (TBD) into a new 50ml centrifuge tube, and then carefully add 25ml of diluted whole blood along the wall without damaging it. The interface between two liquids. Centrifuge at 750g for 25-30 minutes. After centrifugation, aspirate the middle white blood cell layer, wash twice with PBS and count. The initial culture was carried out according to the ratio of 2×10 ⁶ + 3ml NK cell serum-free culture medium + 5% autologous serum + reagent A (ZY-NKZ-0104, IL-21 natural killer cell amplificati-on system). After 6 days of stimulation, culture Perform electrical transfer.

2.Electrotransfection of NK cells with PB transposon system

Prepare the electroporation medium (Celetrix) according to the ratio of liquid A: liquid B = 1:1. The NK cell count is 10 million. Add 100 μl of electroporation medium to resuspend the cells. Add 10 μg of PB transposon plasmid and 5 μg of PB transposase plasmid. Use Celetrix electroporation instrument. The voltage is set to 480V in cell line mode. After electroporation, transfer to culture medium and continue culturing. After 2 days, the electroporation efficiency is measured by flow cytometry.

3. Anti-TRBV12 CAR-NK cell efficiency detection

After electroporation according to the above process, continue to culture for 48 hours to detect the infection efficiency by flow cytometry, and further perform magnetic bead sorting after 14 days of culture. After sorting, flow cytometry is used to detect the infection efficiency. As shown in A and B in Figure 4, the expression efficiency of CAR after sorting is much higher than the positive rate of CAR before sorting, and after continuing to culture for 14 days, the NK cell phenotype was detected and found that the proportion of NK was about 80%, such as Shown in C and D in Figure 4.

Example 5 Detection of CAR-NK killing effect on target cells expressing TRBV12

1. Detection of CAR-NK killing effect on target cells (Jurkat) endogenously expressing TRBV12

In order to compare the ability of (TRBV12-His) CAR-NK cells and (α-TRBV12) CAR-NK cells to eliminate target cells that naturally express TRBV12, we compared the two CAR-NK cells with Jurkat cells at different efficacy-to-target ratios (0.2 :1,0.4:1,0.8:1,1:1) were co-incubated, and the direct killing effect of CAR-NK was detected using a cell lysis experiment, as shown in A in Figure 5; the supernatant cytokines IFN-γ and TNF-α Detection of indirect killing effects is shown in Figure 5, B and C. The results all showed that TRBV12CAR-NK showed stronger killing ability than control NK cells, and (TRBV12-His) CAR-NK had better killing effect on target cells than (α-TRBV12) CAR-NK cells.

2. CAR-NK killing test on target cells overexpressing TRBV12 (CCRF-TRBV12)

Since CCRF cells themselves do not express TRBV12, we first used lentiviral transduction to construct target cells CCRF-TRBV12 that overexpressed TRBV12, and then used different effective target ratios (0.2:1, 0.4:1, 0.8:1, 1:1) Co-incubate, and use cell lysis experiments to detect the direct killing effect of CAR-NK. The supernatant cytokines IFN-γ and TNF-α are used to detect the indirect killing effect, as shown in Figure 6, where A represents the combination of CAR-NK cells and target cells. Lysis of target cells after co-culture with different efficiency-to-target ratios. B and C represent the expression of cytokines TNF-α and IFN-γ in the supernatant. The results all showed that CAR-NK showed stronger killing ability than control NK cells, and (TRBV12-His) CAR-NK was better than (α-TRBV12) CAR-NK cells in killing CCRF-TRBV12 target cells.

The present invention has been disclosed above with preferred embodiments, but they are not intended to limit the present invention. Any technical solutions obtained by adopting equivalent substitutions or equivalent transformations fall within the protection scope of the present invention.

Claims (11)

1. A single-chain antibody targeting TRBV12, characterized by: including a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes CDR-H1, CDR-H2 and CDR-H3. Three complementarity determining regions, the light chain variable region includes three complementarity determining regions composed of CDR-L1, CDR-L2 and CDR-L3; the amino acid sequence of CDR-H1 is shown in SEQ ID NO: 2, and the amino acid sequence of CDR-H2 is as shown in SEQ ID NO: 2. The amino acid sequence is shown in SEQ ID NO:3, the amino acid sequence of CDR-H3 is shown in SEQ ID NO:4; the amino acid sequence of CDR-L1 is shown in SEQ ID NO:5, the amino acid sequence of CDR-L2 is shown in SEQ ID NO :6, and the amino acid sequence of CDR-L3 is shown in SEQ ID NO:7. 2. The single-chain antibody targeting TRBV12 according to claim 1, characterized in that: the full-length amino acid sequence of the antibody is shown in SEQ ID NO: 1. 3. An isolated nucleic acid encoding the single-chain antibody of any one of claims 1-2. 4. The nucleic acid according to claim 3, characterized in that its nucleotide sequence is shown in SEQ ID NO: 8. 5. An expression vector or host cell comprising the isolated nucleic acid of claim 3. 6. A chimeric antigen receptor CAR comprising the single-chain antibody according to any one of claims 1-2, characterized in that: the chimeric antigen receptor CAR further includes a transmembrane domain and costimulatory signaling district. 7. The chimeric antigen receptor CAR according to claim 6, characterized in that: the transmembrane domain is selected from one of CD3ζ, CD8, CD28, NKG2D, 2B4, and DNAM1; or/and The costimulatory signaling region is selected from CD3ζ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD66d, CD2, CD4, CD5, CD28 family, DAP10, DAP12, NKp44, NKG2D, NKp46, NKp30, TNFR family or one or more combinations from the SLAM receptor family. 8. The chimeric antigen receptor CAR according to claim 7, characterized in that: the CD28 family is CD28 or ICOS; the TNFR family is 4-1BB, OX40 or CD27; and the SLAM receptor family is 2B4. 9. A CAR-NK cell expressing the chimeric antigen receptor CAR of claim 6. 10. The preparation method of CAR-NK cells according to claim 9, characterized by comprising the following steps: Isolate and culture NK cells from healthy people; The PB transposon system is used to electroporate NK cells; the PB transposon system expresses anti-TRBV12 CAR; Detect the expression efficiency of CAR in NK cells after electroporation, and further expand the culture. 11. The single-chain antibody according to any one of claims 1-2, the chimeric antigen receptor CAR according to claim 6, and the CAR-NK cell according to claim 9 in the preparation of anti-TRBV12 (+) T cells Application of drugs or products in malignant tumors or TRBV12(+) T cell proliferation diseases.